The experiments described in this proposal are designed to provide insight into the mechanism and regulation of alternative pre-mRNA splicing (AS). The following Specific Aims are proposed. 1. SRp38 and related factors. Analysis of a novel RNA element that blocks in vitro splicing after the first step will be continued. The sequence will be fully defined and its ability to block the second step in a variety of contexts determined. Possible trans-acting regulators, identified by RNA affinity and mass spectrometry (MS), will be characterized. The mechanism of the second-step block, including the possible involvement of known second-step factors, will be elucidated. The possibility that this represents a novel mechanism for regulating AS will be investigated. SRp38 requires a specific coactivator to function as a sequence-specific splicing activator, and efforts to identify and characterize it will be continued. Protein purification and MS have identified the evolutionarily conserved splicing factor Prp5 as a strong candidate. The mechanism by which the coactivator functions will be determined and the possibility that it participates in AS regulation in vivo investigated. 2. Splicing and transcription. Studies to investigate mechanisms by which splicing is functionally coupled to transcription will be continued. In collaboration with R. Roeder, chromatinized templates will be assembled, and the extent and rate of splicing of transcripts produced from these templates determined. Possible effects of specific histone modifications will be investigated. Experiments examining the role of the RNA polymerase II large subunit C- terminal domain (CTD) in splicing will be continued. An ASF/SF2-CTD fusion protein has been used to show that the CTD can enhance splicing in vitro in the presence of a coactivator, which has been purified and tentatively identified by MS as a novel complex between splicing factor U2AF and the Prp19 complex (Prp19C). This complex will be characterized in detail, and its functional significance, especially during spliceosome assembly, determined. The role of U2AF-Prp19C recruitment by the CTD during coupled transcription/splicing will be investigated. 3. Splicing and disease. Experiments to investigate regulation of pyruvate kinase M-form (PKM) AS in cancer will be continued. Recent work has implicated hnRNPA1/A2 and PTB in this process, and additional experiments, including in vivo exon-swapping, RNA binding and in vitro splicing assays, will be performed to determine whether additional proteins participate in PKM splicing control. The physiological significance of these findings will be examined in collaboration with P. Canoll using a rat model for gliablastoma. Retoviral infection will be used to determine whether hnRNPA1/A2/PTB shRNAs can block gliomagenesis, and conversely whether overexpression of these proteins can induce it. Lastly, the function of the Translocated in Liposarcoma (TLS) protein in coupling transcription-splicing will be investigated. Recently identified TLS target genes will be analyzed for changes in AS in TLS knockdown/overexpressing cells, and alterations in recruitment of splicing factors to these genes will be determined. PUBLIC HEALTH RELEVANCE: RELEVANCE: The experiments described in this proposal are designed to increase our understanding of the mechanism and regulation of alternative splicing and its link to transcription. Numerous studies have revealed that changes in alternative splicing occur during development and disease, and the proposed experiments will provide a mechanistic understanding for these changes.